Francisella tularensis is a highly infectious intracellular bacterial pathogen that causes tularemia, a potentially life-threatening disease in humans. Due to the ease of aerosol dissemination of this organism and the minimal inoculum (&#8804;10 bacteria) necessary to cause severe disease, F. tularensis has been categorized as a Category A select agent by the CDC and a priority pathogen by the NIH. In order to cause disease, Francisella subverts and evades multiple arms of the host defense response and co-opts host cells as intracellular replication niches. Francisella[unreadable]s profound manipulation of the host immune response and the novelty of its genome make the study of this fascinating intracellular bacterial pathogen a fertile ground for research very likely to lead to the discovery of novel paradigms and insights into bacterial pathogenesis, immune evasion and host defense. Unfortunately, we still understand relatively little about how Francisella achieves these critical aspects of its pathogenesis. In order to identify genes required for the pathogenesis of Francisella, we performed an unbiased, in vivo, genome-wide negative selection screen in F. novicida, a very closely related BSL-2 level subspecies of Francisella. Our screen was validated by the identification of known virulence genes but most importantly revealed 44 hypothetical genes required for F. novicida virulence, including a novel gene (sirA, or suppressor of inflammatory responses A) that is essential for bacterial virulence in vivo. We demonstrated that an F. novicida mutant strain lacking sirA was strongly attenuated as compared to the wild-type strain following in vivo infection. Interestingly, the sirA mutant was able to enter and replicate within macrophages with wild-type kinetics. However, in vitro macrophage infection experiments revealed that SirA suppresses the activity of TLR2, the innate immune receptor required for initial recognition of Francisella by macrophages and for in vivo host defense. Taken together, our results demonstrate that SirA is a novel, essential mediator of Francisella virulence that plays a critical role in the profound suppression of host defenses by this intracellular pathogen. Elucidating how SirA functions and the breadth of its effects on host defenses is critical not only to understanding Francisella pathogenesis, but to understand strategies likely used by other intracellular pathogens to subvert the immune response. The aims of this proposal are to identify the TLR2-dependent defenses suppressed by the novel Francisella virulence factor SirA, the level in the TLR2 pathway at which SirA acts, the effect this suppression has on in vivo infection, and to perform a detailed molecular characterization of this novel protein.